Hepatoselective pharmaceutical actives

ABSTRACT

The invention provides an analogue of a pharmaceutical active whose molecular weight is less than 25,000 Daltons, the analogue comprising a pharmaceutical active whose molecular weight is less than 25,000 Daltons covalently linked to a pendant molecular group wherein as a result of the administration of the composition to the human or animal body an active complex having a molecular weight of 25,000 Daltons or greater is present in the human or animal circulatory system. The analogue is hepatoselective when administered to the circulatory system. Preferably the analogue is an insulin analogue comprising an insulin or functional equivalent thereof covalently linked to the pendant molecular group wherein the active complex is an insulin complex. Such an insulin analogue may be used in a method of insulin replacement therapy.

This application is a REI of Ser. No. 09/097,535 filed Jun. 15, 1998;U.S. Pat. No. 6,063,761 which is a divisional of Ser. No. 08/596,285filed Feb. 13, 1996; U.S. Pat. No. 5,854,208 which is a 371 ofPCT/GB94/01784 filed Aug. 15, 1994.

The present invention relates to novel hepatoselective pharmaceuticalactives. In particular it relates to novel hepatoselective insulinanalogues suitable for use in an improved treatment of diabetesmellitus.

When drugs and other pharmaceutical actives are administered to thehuman or animal body it may be required that the active is needed to bepresent primarily in the liver or to act primarily on the tissues of theliver. That is, the active is required to be hepatoselective. Achievinghepatoselectivity can be difficult, in particular where the active isadministered by injection into the skin. One case in which achievementof hepatoselectivity would be especially desirable is the administrationof insulin.

The hormone insulin, secreted by the pancreas, has various importantroles to play in glucose metabolism. In the liver, after binding to cellsurface receptors, insulin promotes the conversion of glucose toglycogen (glycogenesis), promotes protein synthesis and inhibits fatbreakdown. Insulin deficiency results in breakdown of glycogen(glycogenolysis), protein and conversion of products of fat and proteinbreakdown to glucose (gluconeogenesis) leading to a raised plasmaglucose level (hyperglycaemia). In subjects who produce adequate amountsof insulin the blood glucose level remains within a certain range. Anyexcess of glucose is stored in the liver and muscles as glycogen.

Insulin also acts on cell membrane receptors in other tissues to enhancethe entry of glucose into cells, thereby diminishing the plasma glucoseconcentration.

Thus insulin acts to reduce the plasma glucose level by reducing theproduction of glucose by the liver and by increasing uptake andmetabolism of glucose by the liver and by increasing uptake andmetabolism of glucose by peripheral tissues.

Deficiency of insulin due to disease of the islets of Langerhans and/ordeficiency of insulin action results in diabetes mellitus, a conditionin which the blood glucose concentration is high.

In subjects who are not diabetic insulin is produced in the pancreas andtransported directly to the hepatic circulation and hence is transportedto the liver before any other organs or regions of the body. Thus theliver experiences a very high exposure to the insulin produced. Usuallyat least 50% of the insulin produced is bound to receptors in the liver,and hence acts in the liver. Insulin bound to receptors in the liver isremoved from the circulation and degraded by the liver cells. Theinsulin which is not bound in the liver and hence passes to theperipheral circulation is therefore at a much lover concentration. Thusthe peripheral tissues (eg fat and muscle) which are also targets of theinsulin experience a smaller exposure to the insulin secreted.

In diabetic subjects treatment is often carried out byinsulin-replacement therapy. In general an insulin preparation isinjected subcutaneously. The most common subcutaneous insulin regimeninvolves twice-daily injection of mixtures of short- andintermediate-acting insulin preparations. Insulin is absorbed from thesubcutis into the peripheral circulation and thence to the entire body,including the liver. With such a system the liver and the peripheraltissues tend to experience approximately equal exposure to insulin.

There are various disadvantages and negative side-effects with the useof this system.

First, if sufficient insulin is injected to enable a high enoughconcentration to be present in the hepatic circulation then too high aconcentration will be present in the peripheral circulation. Similarlyif a suitable concentration is present in the peripheral circulation theconcentration of insulin in the hepatic circulation will be inadequate.

There is believed to be a danger associated with high concentrations ofinsulin in the peripheral circulation (hyperinsulinaemia) ofcardiovascular disease.

Second, there is a serious risk of hypoglycaeaia in diabetic subjectsreceiving insulin replacement therapy by subcutaneous injection. Aconcentration of insulin in the peripheral circulation which is too highcan lead to a blood sugar level which is too low and subsequentcollapse.

It would therefore be desirable to be able to direct a largeconcentration of insulin directly to the hepatic insulin receptors witha lower concentration of insulin being directed to the peripheralinsulin receptors. It would also be desirable to achieve suchhepatoselectivity for other active molecules.

Some attempts have been made to obtain the desired hepatoselectivity ofinsulin. Intravenous injection directly into the hepatic portalcirculation could allow injected insulin to pass directly to the liverbefore reaching the peripheral circulation. Such a system is unsuitablefor general use due to the difficulty and complicated nature of itsoperation; in particular it is not suitable for use by diabetic patientsthemselves except under exceptional circumstances.

An attempt has been made to render insulin hepatoselective by injectingit subcutaneously whilst encapsulated in lipid vesicles. (Spangler,Ronald S., “Selective Insulinisation of Liver in Conscious DiabeticDogs”, Am. J. Physiol. 249 (Endocrinol. Metab. 12): E152-E159, 1985).Insulin encapsulated in lipid vesicles (designated vesicle encapsulatedinsulin VEI) was targeted to hepatocytes by means of a digalactosyldiglyceride moiety incorporated on the outside of the lipid vesicles.Using this approach it was possible to alter the distribution of anadministered glucose load to favour hepatic deposition.

The present inventors have found (British Diabetic Association Medicaland Scientific section Conference, Manchester, Apr. 13-15, 1989) thatcovalent dimers of insulin (molecular weight ±12,000 Daltons rather than±6,000 for insulin monomer) have a greater effect on hepatic glucoseoutput than on peripheral uptake and utilisation. That is, the covalentinsulin dimers appear to act preferentially on hepatocytes rather thanon cells of the peripheral tissues. It has also been found thatproinsulin shows this hepatoslectivity to a smaller degree.; Proinsulinis the zymogen which is cleaved to form the active hormone insulin. Theproinsulin molecule is larger than the active insulin molecule.

The cells of the peripheral tissues, for instance fat and muscle, areseparated from the blood vessels by the capillary endothelium. In theliver however there is no such barrier between blood vessels andhepatocytes.

It is thought that transport across the capillary endothelium is mainlyby diffusion i.e. active transport of insulin across the capillaryendothelium does not occur to any significant extent. Therefore thepresent inventors believe that the absorption of insulin into theperipheral tissues is determined by factors influencing diffusivetransport, in particular by steric hindrance or size of molecules. Thisis believed to be a reason for the relative hepatoselectivity ofcovalent insulin dimers and proinsulin when compared with insulinmonomers; both have free access to hepatocytes but the larger covalentinsulin dimer or proinsulin is absorbed into the peripheral tissues fromthe bloodstream more slowly than is the insulin monomer. Thus the largermolecules spend a longer time in the bloodstream before being absorbedinto the peripheral tissues and are thus are more likely to reach theliver, where they may be active more easily.

According to the present invention there is provided the use of ananalogue of a pharmaceutical active whose molecular weight is less than25,000 Daltons in the manufacture of a composition for use in a methodof treatment of the human or animal body, the analogue comprising apharmaceutical active whose molecular weight is less than 25,000 Daltonscovalently linked to a pendant molecular group wherein as a result ofthe administration of the composition to the human or animal body anactive complex having a molecular weight of 25,000 Daltons or greater ispresent in the human or animal circulatory system.

The invention is of particular usefulness when the analogue is aninsulin analogue which comprises an insulin or functional equivalentthereof covalently linked to a pendant molecular group, so that as aresult of the administration of the composition to the human or animalbody an insulin complex having a molecular weight of 25,000 Daltons orgreater is present in the human or animal circulatory system. Thisinvention is also applicable to other pharmaceutical actives ofmolecular weight below 25,000 Daltons which are administered so as toenter the circulatory system and which are required to behepatoselective and for which similar problems as those encountered withthe administration of insulin therefore also apply. In thisspecification the invention is discussed primarily in terms of its usefor giving hepatoselectivity to insulin or a functional equivalentthereof, but it will be understood that the disclosures made are equallyapplicable to other pharmaceutical actives of molecular weight less than25,000 Daltons.

According to a first, particularly preferred, embodiment of theinvention the pendant molecular group has an affinity for one or morebinding proteins present in the human or animal circulatory systemwherein the total molecular weight of the insulin or functionalequivalent thereof (or other pharmaceutical active) and the additionalmolecular group and the one or more binding proteins is 25,000 Daltonsor greater.

Binding proteins “in the human or animal circulatory system” are thosewhich are present in the blood plasma.

Thus when the analogue is introduced into the bloodstream the one ormore binding proteins will become non-covalently linked to theadditional molecular group, forming a complex which has a molecularweight of 25,000 Daltons or greater.

According to a second embodiment of the invention an insulin analogue(or analogue of another pharmaceutical active) is used whichadditionally comprises one or more binding proteins non-covalentlyattached to the pendant molecular group wherein the total molecularweight of the insulin or functional equivalent thereof (or otherpharmaceutical active) and the pendant molecular group and the one ormore binding proteins is 25,000 or greater.

In this second embodiment the analogue is a complex having a molecularweight of 25,000 or greater and may be administered into the circulatorysystem as such, for instance intravenously.

According to a third embodiment of the invention an insulin analogue (oranalogue of other pharmaceutical active) is used in which the totalmolecular weight of the insulin or functional equivalent thereof (orother pharmaceutical active) and the covalently linked pendant moleculargroup is 25,000 Daltons or greater.

As with the second embodiment, the analogue is a complex having amolecular weight of 25,000 or greater and may be administered into thecirculatory system as such, for instance intravenously.

For use in the first aspect of the present invention there is providedan analogue of a pharmaceutical active whose molecular weight is lessthan 25,000 Daltons comprising a pharmaceutical active whose molecularweight is less than 25,000 Daltons covalently linked to a pendantmolecular group, said pendant molecular group having an affinity for oneor more binding proteins present in the human or animal circulatorysystem.

As explained above, the invention is especially useful when the compoundis an insulin analogue comprising an insulin or functional equivalentthereof covalently linked to a pendant molecular group which has anaffinity for one or more binding proteins present in the human or animalcirculatory system.

Such an insulin analogue (or analogue of other pharmaceutical active)may be injected subcutaneously and absorbed into the bloodstream throughthe capillary endothelium without difficulty. When in the bloodstreamthe insulin analogue will come into contact with the binding protein forwhich the covalently-linked pendant molecular group has an affinity.Thus at least some molecules of the insulin analogue will become boundto the said binding protein, forming an insulin complex. Bindingproteins tend to be bulky molecules of high molecular weight. Theytherefore tend not to diffuse out through the capillary endotheliumeasily and remain in the bloodstream. Thus the effective size ofmolecule and hence the molecular weight of the bound insulin analogue isincreased dramatically. Absorption from the blood vessels into theperipheral tissues, for instance fat and muscle, through the capillaryendothelium is now greatly inhibited due to the attachment of theinsulin analogue to the high molecular weight binding protein. In theliver however there is no such barrier therefore the insulin analogueeven with the associated binding protein may have access to thehepatocyte insulin receptors essentially to the same degree as wouldconventional insulin.

In the same way an analogue of another pharmaceutical active may beinduced not to diffuse out through the capillary endothelium and toremain in the bloodstream until it is carried to the liver where it mayact or be taken up as required.

Binding of the pendant molecular group and the binding protein is notcovalent. Binding forces may be for instance electrostatic (egattraction of opposite charges, hydrogen bonding) or hydrophobic. Thusbinding is not permanent. The insulin analogue (or analogue of otherpharmaceutical active) may be absorbed onto the hepatic tissues in itsbound form. Molecules of the insulin analogue which have not been boundby the binding protein or which have become bound and subsequentlyunbound are capable of passing through the capillary endothelium intothe peripheral tissues.

The covalently-linked molecular group is attached in such a way that theactive site (or sites) of the insulin equivalent or other pharmaceuticalactive remains available to carry out its prescribed functions.

Attachment of a suitable molecular group to the insulin equivalent (orother active) may be carried out by conventional chemical methods knownto those skilled in the art.

According to this aspect of the invention there is also provided amethod of insulin replacement therapy comprising subcutaneous injectionof a preparation comprising an insulin analogue as described above.

Preferred features of the first embodiment of the invention will now bedescribed in detail.

The insulin or functional equivalent thereof, when, insulin is theactive used, may be any of the insulins conventionally used in insulinreplacement therapy.

J. Brange et al (“Monomeric Insulins and their Experimental and ClinicalImplications”, Diabetes Care, vol. 13, no. 9, September 1990) and othershave studied the possibility of developing insulins with reducedtendencies to self-association. These insulins are absorbed from thesubcutis into the bloodstream more rapidly than is the form in whichinsulin is usually found in pharmaceutical formulations. Insulin assumesan associated state in pharmaceutical formulation. Six monomers ofinsulin associate to form hexamers. The association is non-covalent.With the use of DNA technology Brange et al and others have preparedinsulins which remain dimeric or even monomeric at high (pharmaceutical)concentration by the introduction of one or a few amino acidsubstitutions into human insulin. Insulins with reduced associationcapacity as described by Brange et al and others are preferred for useas the insulin equivalent to which the additional molecular group isattached. When the pharmaceutical active is an insulin equivalent suchinsulins are preferred, because their reduced tendency to self-associatemeans that they are more rapidly absorbed into the bloodstream from thesubcutis than are conventional insulins which tend to be injected inhexameric form.

Insulins of this type have also been developed by Eli Lilly. These aredescribed in Protein Engineering, Vol 5, 519-525 and 527-533 (1992)(both D. N. Brems et al). Studies of amino acid modified monomericinsulins have also been described in Diabetes 40 Suppl. 1 (1991), 423A(Howey et al) and 464A (Shaw et al).

The pendant molecular group covalently linked to the insulin equivalentor other active may be any molecular group which has an affinity for abinding protein present in the circulatory system and which will notitself act in the body to give detrimental effects. The molecular groupchosen should usually be of a molecular weight similar to or less thanthat of the insulin equivalent or other active. Preferably the totalmolecular weight of the insulin or functional equivalent thereof orother active and the additional molecular group is less than 25,000,more preferably less than 20,000 or 15,000, and may be less than 12,000.This is in order that attachment of the pendant molecular group to theinsulin equivalent or other active to form the analogue should nothinder the passage by diffusion of the injected analogue from thesubcutis through the capillary endothelium into the bloodstream.Preferred molecular groups are molecular structures which possess anaffinity for one or more proteins which are naturally present in thecirculation. They may be based on naturally-occurring hormones or onfunctional equivalents of such hormones which also possess affinity totheir binding proteins or they may be based on other substances forwhich such binding proteins exist. The pendant molecular group should beharmless when injected into the body; this may be achieved for instanceby ensuring that the concentration of insulin analogue or analogue ofother active in the bloodstream is high enough to allow the beneficialeffects of insulin or other therapy to be felt, but low enough toprevent any effects which might be due to the pendant molecular groupbeing felt, or by ensuring that the insulin analogue or analogue ofother active is not present in parts of the body where the pendantmolecular group might be active, or by rendering the pendant moleculargroup inactive by structural modification which nevertheless preservesits ability to bind to its binding protein.

The pendant molecular group may be itself an insulin equivalent, forinstance insulin-like growth factor 1 (IGF1). This polypeptide has anaffinity for IGF1 binding proteins, which circulate naturally in thehuman bloodstream.

The pendant molecular group may be a native or modified thyroxyl group,derived from the human thyroid hormone thyroxine3,5,3′,5′-L-tetraiodothyronine (T4). There are several binding proteinspresent in the human circulatory system which have an affinity for theT4 group, for instance thyroxine binding globulin (TBG), thyroxinebinding prealbumin (TBPA) and albumin. These proteins are knowncollectively as thyroxine binding proteins (TBP).

Other suitable groups derived from hormones or their functionalequivalents may be used. Suitable groups may be ascertained by a skilledperson using methods known in the pharmaceutical field.

The pendant molecular group with an affinity for a binding protein maybe covalently attached directly to the insulin equivalent (or otheractive), as explained above in a place chosen so that the additionalmolecular group does not inhibit the action of the insulin equivalent.The structure of insulin and the locations of its active sites are wellknown, therefore when insulin is used those skilled in the art will beable to establish appropriate positions at which to attach the saidmolecular group so as not to interfere significantly with the action ofthe insulin or equivalent.

Alternatively a short molecular chain (or “spacer arm”) may be used tolink the insulin equivalent (or other active) and the said moleculargroup. The spacer arm is linked covalently both to the insulinequivalent and to the pendant molecular group. Such a spacer arm ensuresthat the insulin equivalent and binding protein are distanced from oneanother thus preventing substantial interference with insulin activityby the (usually bulky) high molecular weight binding protein. The spacerarm is usually a linear chain, preferably of from 3-10 carbon atoms inlength. Such a spacer arm may be for instance an aminohexanoyl (AH)group, which is a six-carbon chain. Other groups may of course be usedas a spacer arm. For example aminoacids either singly or linked as smallpeptide sequences could be used.

It should be ensured that the binding protein which has an affinity forthe pendant molecular group is present in the blood plasma insufficiently large amounts that the trapping of the binding protein bythe insulin analogue (or analogue of other active) with the covalentlylinked molecular group will not deplete the levels of binding protein inthe blood to detrimental effect. For instance the level of bindingprotein should not be depleted so that insufficient binding protein isavailable to carry out its usual function in the body, if any.

The insulin analogue (or analogue of other active) of the invention maybe produced by various methods, for instance: chemical reaction of theinsulin or insulin equivalent or other active with a substance orsubstances which include the pendant molecular group; protein synthesisof the complete analogue; production by a genetically engineered microorganism.

The insulin analogue of the invention may be used in a method of insulinreplacement therapy.

An insulin analogue according to the invention or a mixture of two ormore different insulin analogues according to the invention form part ofan insulin preparation. This insulin preparation is suitable for use ina method of treatment of the human or animal body, preferably suitablefor subcutaneous injection, and may comprise a treatment for diabetes.Additional ingredients may be added which modify the rate of absorptionfrom the subcutaneous depot into the circulation. The insulin-basedpreparation preferably is suitable for subcutaneous injection, in whichcase it is therefore suitable for use by sufferers from diabetes onthemselves.

The insulin preparation may comprise the hepatoselective insulinanalogue of the invention and a conventional non-hepatoselectiveinsulin. On subcutaneous injection and passing into the bloodstream theconventional insulin will act in the peripheral tissues whilst theinsulin analogue of the invention provides a controlled, hepatoselectiveaction.

The analogues of other pharmaceutical actives of the invention may alsobe used in methods of treatment or therapy by subcutaneous injection ofa preparation comprising the analogue of the active.

According to the second embodiment of the invention an insulin analogue(or analogue of other active) with a covalently attached pendantmolecular group is used with a binding protein already non-covalentlylinked to the pendant molecular group. A composition comprising such aninsulin analogue would be more suitable for intravenous administrationthan for subcutaneous administration due to the very high molecularweight of the insulin analogue in the composition (as compared with theinsulin analogue of the first embodiment, which forms an insulin complexof molecular weight greater than 25,000 only after administration intothe circulatory system). It may be desirable in some cases however toprovide such an insulin analogue or analogue of other active for use inthe manufacture of a composition suitable for intravenousadministration.

The pendant molecular group and binding proteins may be any of thosedescribed above as suitable for the first embodiment of the invention.In addition the binding proteins may be proteins which do not occurnaturally in the human or animal circulatory system (and which areharmless when introduced into the circulatory system) and the pendantmolecular group may be any group with an affinity to such a protein.

According to a third embodiment of the invention an insulin analogue (oranalogue of other active) is used which comprises an insulin orfunctional equivalent thereof (or other active) with a large groupcovalently attached. Such a group should be large enough that the entireinsulin analogue has a molecular weight of at least 25,000. It shouldalso, like the pendant molecular group of the first embodiment of theinvention, be harmless when introduced into the body. This may beachieved as described above for the pendant molecular group. The largemolecular group of the third embodiment of the invention may forinstance be based on a polypeptide structure or on other suitablepolymeric structures.

The analogues of the second and third embodiments of the invention maybe produced by the methods mentioned above as suitable for theproduction of the analogue of the first embodiment of the invention. Forinstance suitable methods in the case of the analogue of the thirdembodiment include chemical reaction of the insulin or equivalent orother active with a substance of substances which include the covalentlylinked molecular group, protein synthesis of the complete analogue andproduction by a genetically engineered micro organism. In the case ofthe second embodiment the section of the analogue comprising the insulinor equivalent or other active and the additional molecular group may besynthesized in the same way as may be the insulin of the firstembodiment. The binding protein may be synthesized for instance byprotein synthesis, production by a genetically engineered microorganism, extraction from a naturally-occurring organism. Non-covalentattachment of the binding protein or proteins to the pendant moleculargroup may be achieved by methods known in the art.

The analogues of the second and third aspects of the invention aresuitable for incorporation in compositions which are to be administeredintravenously. Where the active is insulin or a functional equivalentthereof, such compositions may be used in a method of insulinreplacement therapy. Such compositions comprise one or more insulinanalogues of the second and third embodiments of the invention. Thecomposition may also comprise additional ingredients which may be chosenby those skilled in the art.

EXAMPLES

The present inventors have undertaken a study to explore the possibilitythat insulin analogues with restricted access to peripheral tissues maydisplay relative hepatoselectivity in vivo.

Analogues of insulin which include a thyroxyl moiety which binds tothyroid hormone binding protein (TBP) have been designed and tested.N^(αB1)-thyroxyl-insulin (T4-Ins) and N^(αB1)-thyroxyl-aminohexanoylinsulin (T4-AH-Ins) were synthesized using methods of chemical synthesisgiven below.

Preparation of thyroxyl insulins

Abbreviations:

Msc=methylsulphonylethyloxycarbonyl

Boc=tert.butyloxycarbonyl

DMF=dimethylformamide

DMSO=dimethylsulfoxide

mp=melting point

ONSU=N-oxysuccinimide ester

Example 1

B1-thyroxyl-insulin (porcine) (T4-Ins)

Protected thyroxine derivatives:

Msc-L-thyroxine (I)

776 mg (1 mmol) L-thyroxine in 2 ml dimethylsulfoxide was reacted with530 mg (2 mmol) Msc-ONSu in the presence of 139 μl (1 mmol)triethylamine at room temperature for 18 hours. After concentration invacuo the oily residue was taken up in ethyl acetate, the organic layerwashed with water, dried over Na₂SO₄, and the solvent removed in vacuo.The solid residue was crystallised from hexane.

Yield: 651 mg (70.2% of theory),

mp: 200° C.

Msc-L-thyroxine-N-oxysuccinimide ester (II)

To a cooled (0° C.) solution of 371 mg (0.4 mmol) of Msc-thyroxine and40 mg (0.4 mmol) N-hydroxysuccinimide in 1 ml tetrahydrofurane aprecooled solution of 82.5 mg N,N′-dicyclohexycarbodiimide in 0.5 mltetrahydrofurane was added under stirring. After further stirring for 3hours the precipitate was removed by filtration, and the filtrateconcentrated in vacuo. Crystallisation of the solid residue frommethylenechloride/petroleum ether.

Yield: 295 mg (71%, based on I)

mp: 180° C.

B1-thyroxyl-insulin (porcine) (III)

To a solution of 100 mg (approx 0.016 mmol) A1,B29-Msc₂-insulin(prepared according to Schüttler and Brandenburg, Hoppe-Seyler's Z.Physiol, Chem. 360, 1721-1725 (1979)) and 18 μl (0.160 mmol)N-methylmorpholine in 2 ml of DMSO 116.4 ml (0.160 mmol) of II in 0.2 mlDMSO were added. After stirring for 6 h at room temperature the insulinderivative was precipitated with ether/methanol (9:1, v/v), isolated bycentrifugation, washed 3 times with ether/methanol, and dried in vacuo.

Msc groups were removed by treatment with NaOH/dioxane/methanol at 0°C., and III was purified by gel filtration on Sephadex G 50 fine asdescribed (Geiger et al, Chem. Ber. 108, 2758-2763 (1975)).Lyophilization gave 78.4 mg B1-thyroxyl-insulin (75%, based onMsc₂-insulin).

Example 2

B1-thyroxyl-aminohexanol-insulin (porcine) (IV) (T4-AH-Ins)

1. BOC-ε-aminohexanoic acid was prepared by reacting 1.32 g (10 mmol)ε-aminohexanoic acid with 2.4 g (11 mmol) di-tert.butyldicarbonate indioxane/water at pH 9 and was obtained in 87% yield (2.2 g). Mp afterrecrystallization from ethyl acetate: 75° C.

2. 22.8 mg (0.096 mmol) Boc-aminohexanoic acid was preactivated with 13mg 1-hydroxybenzotriazole and 17.8 mg (0.09 mmol) dicyclohexylcarbodiimide in 0.7 ml dimethylformamide for 1 h at 0° C. and 1 furtherhour at room temperature.

3. Then, a solution of 100 mg (approx. 0.016 mol) A1,B29-Msc₂-insulinand 18 μl (0.160 mmol) N-methylmorpholine in 1 ml of DMF was added.After stirring for 70 minutes at room temperature the mixtures wasfiltered, and the insulin derivative was precipitated with ethylether/methanol (9:1, v/v), isolated by centrifugation, washed 3 timeswith ether/methanol, and dried in vacuo.

4. The Boc protecting group was cleaved by treatment of the product with3 ml trifluoro acetic acid for 1 hour at room temperature. The solutionwas concentrated in vacuo, the insulin derivative precipitated withether, isolated, washed with ester, and dried. Yield: 77.8 mg ofB1-aminohexyl-A1,B29-Msc₂-insulin.

5. 102 mg (0.016 mmol) of this derivative was dissolved in 2 mldimethylformamide and 18 μl (0.16 mmol) N-methylmorpholine. Afteraddition of 116 mg (0.16 mmol) Msc-thyroxine-N-oxysuccinimide ester in0.2 ml dimethylformamide the mixture was stirred for 3 hours at roomtemperature. The protected insulin was isolated by precipitation withmethanol/ether and isolated as described above.

6. Msc groups were removed by treatment with NaOH/dioxane/methanol at 0°C., and IV was purified by gel filtration on Sephadex G 50 fine asdescribed (Geiger et al, op. cit.). Lyophilization gave 39.1 mgB1-thyroxylaminohexanoyl-insulin (37%, based onaminohexyl-Msc₂-insulin).

T4-Ins, T4-AH-Ins and insulin (Ins) were infused into four anaesthetizedbeagles with D-³H-3-glucose for measurement of the rates of glucoseproduction (Ra) and glucose disposal (Rd). Euglycaemia and glucosespecific activity were maintained by variable infusion of D-glucose withD-³H-3-glucose.

With all three materials glucose Rd was increased and glucose Radecreased from basal level 2.70±0.19 mg. kg⁻¹ min⁻¹, (p<0.05). In eachexperiment insulin-like activity for Ra and Rd was calculated as thearea between the basal values of each of these variables and subsequentvalues plotted graphically against time (AUC). For Ins, T4-Ins andT4-AH-Ins respectively, AUC for Ra values were −431±121, −226±154 and−357±50 (mean±SEM, mg/kg), (no significant differences) and AUC for Rdvalues were 1142±160, 629±125 and 830±178 mg/kg, both analoguesdifferent from Ins p<0.05.

These results indicate that insulin analogues of the invention act to agreater extent on the tissues of the liver than on those of theperipheral regions of the body, such as fat and muscle.

1. A method of treating diabetes in a human or an animal, comprising thestep of: administering a conjugate compound having a molecular weight ofless than 25 kD comprised of a pharmaceutically active compound and apendant molecular moiety which is a naturally occurring hormone or ananalogue thereof, wherein the pendant molecular moiety has an affinityfor one or more binding proteins naturally present in the circulatorysystem of a human or an animal whereby the conjugate reaches thesystemic circulation of the human or the animal and in the circulationforms a complex having a molecular weight of more than 25 kD with theone or more binding proteins.
 2. A treatment method according to claim1, wherein the pharmaceutically active compound is insulin or afunctional equivalent of insulin.
 3. A treatment method according toclaim 1, wherein the pendant molecular moiety is a thyroid hormone.
 4. Atreatment method according to claim 3, wherein the thyroid hormone isthyroxine.
 5. A treatment method according to claim 1, wherein thependant molecular moiety if IGF1.
 6. A treatment method according toclaim 2, wherein the pendant molecular moiety is a thyroid hormone.
 7. Atreatment method according to claim 6, wherein the thyroid hormone isthyroxine.
 8. A treatment method according to claim 1, wherein thependant molecular moiety is linked to the pharmaceutically activecompound by a spacer.
 9. A treatment method according to claim 8,wherein the spacer is a linear chain of from 3 to 10 carbon atoms inlength.
 10. A treatment method according to claim 1, wherein the bindingprotein is selected from the group consisting of albumin,thyroxine-binding pre-albumin, thyroxine-binding globulin, andcombinations of those proteins.
 11. A treatment method according toclaim 2, wherein the binding protein is selected from the groupconsisting of albumin, thyroxine-binding pre-albumin, thyroxine-bindingglobulin, and combinations of those proteins.
 12. A method according toclaim 7, wherein the binding protein is selected from the groupconsisting of albumin, thyroxine-binding pre-albumin, thyroxine-bindingglobulin, and combinations of those proteins.
 13. A method of deliveringa pharmaceutically active compound to a human or an animal comprising:a) covalently bonding to the pharmaceutically active compound a pendantmolecular moiety which is a naturally occurring hormone or an analoguethereof to form a conjugate compound which has a molecular weight ofless than 25 kD; and b) administering the said conjugate compound to ahuman or an animal whereby the conjugate compound is delivered to thecirculatory system of the said human or animal and forms a complextherein with one or more binding proteins naturally present in the saidcirculatory system, said complex having a molecular weight of more than25 kD.
 14. A method of directing a pharmaceutically active compoundselectively to the liver of a human or animal comprising the steps: a)covalently bonding to the pharmaceutically active compound a pendantmolecular moiety which is a naturally occurring hormone or an analoguethereof to form a conjugate compound which has a molecular weight ofless than 25 kD; and b) administering the said conjugate compound to ahuman or an animal whereby the conjugate compound is delivered to thecirculatory system of the said human or animal and forms a complextherein with one or more binding proteins naturally present in the saidcirculatory system, said complex having a molecular weight of more than25 kD.
 15. A method of forming a complex in the systemic circulation ofa human or animal comprising the Steps of: a) covalently bonding to thepharmaceutically active compound a pendant molecular moiety which is anaturally occurring hormone or an analogue thereof to form a conjugatecompound which has a molecular weight of less than 25 kD; and b)administering the said conjugate compound to a human or an animalwhereby the conjugate compound is delivered to the circulatory system ofthe said human or animal and forms a complex therein with one or morebinding proteins naturally present in the said circulatory system, saidcomplex having a molecular weight of more than 25 kD.
 16. A methodaccording to claim 2 in which the pendant molecular moiety is conjugatedat the alpha amino group of the B1 residue of the insulin molecule. 17.A method according to claim 6 wherein the pendant molecular moiety isconjugated at the alpha amino group of the B1 residue of the insulinmolecule.
 18. A method of treating diabetes in a human or animal withinsulin, or a functional equivalent thereof, comprising the step ofadministering a conjugate compound having a molecular weight of lessthan 25 kD comprised of insulin, or a functional equivalent of insulin,covalently bound to a molecule having an affinity for one or morebinding proteins naturally present in blood plasma of a human or animal,whereby the conjugate reaches the systemic circulation of the human orthe animal and in the circulation forms a complex having a molecularweight of more than 25 kD with the one or more binding proteins.
 19. Amethod of selectively delivering a pharmaceutically active compound tohepatocytes in a human or animal comprising (a) covalently bonding tothe pharmaceutically active compound a pendant molecular moiety to forma conjugate compound which has a molecular weight of less than 25 kD and(b) administering said conjugate compound to a human or an animalwhereby said conjugate compound is delivered to the circulatory systemof the said human or animal and forms a complex therein with one or morebinding proteins naturally present in said circulatory system, saidcomplex having a molecular weight of more than 25 kD.
 20. A method asclaimed in claim 19 wherein the pharmaceutically active compound isinsulin or a functional equivalent thereof.